(1) Field of the Invention
The present invention relates to diagnostic biomarkers. In particular, the present invention relates to biomarkers of aging and disease in the retina.
(2) Description of Related Art
Age-related macular degeneration (AMD) is a disease leading to severe visual loss and legal blindness in the elderly population (Klein et al. 1992; Mitchell et al. 1995). The pathophysiology of AMD is complex and can include genetic predispositions, accumulation of lipofuscin and drusen, local inflammation and neovascularization. Recently four independent research groups used different methods to screen the genomes from different groups of AMD patients. All four studies discovered a commonly inherited variant (Y402H) of the complement factor H (CFH) gene that significantly increases the risk of AMD (Edwards et al. 2005; Hageman et al. 2005; Haines et al. 2005; Klein et al. 2005). This finding links genetics and inflammation. Before this finding, the study of the components of drusen had provided compelling evidence that inflammatory and immune-mediated events participate in the development of drusen and progression of AMD. Protein components of drusen include immunoglobulins, components of the complement pathway (e.g., C5 and C5b-9), molecules involved in the acute-phase response to inflammation (e.g., Amyloid P component), and proteins that modulate the immune response (e.g., vitronectin, clusterin, and apolipoprotein E) (Hageman and Muffins 1999; Hageman et al. 1999; Johnson et al. 2000; Mullins et al. 2000). The finding that macrophages are important in choroidal neovascularization (CNV) also supports the involvement of inflammation in AMD (Grossnikiaus et al. 2002). Recent research provided further evidence that inflammation is involved in the development of AMD (Chen et al. 2007; Laine et al. 2007; Schaumberg et al. 2007; Skerka et al. 2007) and the link between inflammation, drusen and oxidative stress (Wu et al. 2007; Hollyfield et al. 2008; Wang et al. 2008).
During inflammation, large fluxes of nitric oxide (NO) are released through the activation of inducible nitric oxide synthase (Marletta et al. 1988; Carreras et al. 1994). Nitrite concentration is reported to be nearly doubled in the diabetic retina (El-Remessy et al. 2003). Cigarette smoking, which has been strongly associated with the development of AMD (Solberg et al. 1998), is also an important chronic contributor to human NO exposure (Council 1986; Borland and Higenbottam 1987). Patients with AMD have significantly higher plasma NO levels than control subjects (Evereklioglu et al. 2003). NO itself is a relatively unreactive radical, however, it is able to form other reactive intermediates including nitrite (NO2−), peroxynitrite (ONOO−), NO2, and N2O3, etc that can modify proteins, lipids and other compounds. Nitrite is one of the major NO metabolic products and has been used as a marker of NO production (Farrell et al. 1992; Gaston et al. 1993). In addition, nonenzymatic nitration of long lived protein such as extracellular matrix proteins is a well known pathway that has been associated with inflammation (Bailey et al. 1998; Paik et al. 2001). The extracellular matrix proteins, such as collagen and elastin have been reported to be nonenzymatically modified by nitrite at physiological pH (Paik et al. 1997; Paik et al. 2001). Applicants have shown that nitrite-modification of basement membrane-like extracellular matrix proteins can impart deleterious effects on adjacent epithelial cell function and viability (Wang et al. 2005) and impair phagocytic capacity (Sun et al. 2007).
Bruch's membrane lies between the choroidal capillary bed and retinal pigment epithelial (RPE) cells. The exchange of various materials between the underlying choriocapillaris and overlying RPE occurs through Bruch's membrane (Lyda et al. 1957; Sellner 1986). Bruch's membrane is permeable to macromolecules up to 300 kD in size in healthy eyes, but there are numerous examples of pathological processes in which larger macromolecules or even cells, including macrophages and leukocytes, can traverse Bruch's membrane in the diseased eye (Crane and Liversidge 2008). In addition to Bruch's membrane, trafficking of material from the RPE to the choriocapillaris is limited in the healthy eye by tight junctions between adjacent cells of the RPE monolayer. This outer blood-retinal barrier is part of the specialized ocular microenvironment that confers protection or immune privilege to mitigate the effect of deleterious immune responses (Streilein 2003). Nevertheless, this barrier is altered in pathological circumstances, and breakdown of the outer blood retinal barrier, including macrophage and leukocyte infiltration of the retina, are implicated in many diseases including AMD (Jha et al. 2007). Several investigators have suggested that age-related damage to Bruch's membrane allows for the accumulation of abnormal extracellular deposits, called drusen, between the basal lamina of the RPE and the inner collagen layer of Bruch's membrane (Newsome et al. 1987; Pauleikhoff et al. 1990; Mullins et al. 2000; Crabb et al. 2002). The accumulation of drusen is thought to elicit a local inflammatory response (Anderson et al. 2002; Yasukawa et al. 2007; Hollyfield et al. 2008).
Recently Applicants have shown that age-related changes in human Bruch's membrane can exert significant deleterious effects on RPE function that are independent of cell aging, including impairing the ability of cultured RPE to phagocytize photoreceptor outer segments (Sun et al. 2007). A similar effect on RPE function is observed after nonenzymatic nitration of RPE basement membrane in tissue culture (Wang et al. 2005). Surprisingly, there have been no studies that have reported nitrite modification occurring in intrinsic Bruch's membrane proteins or extrinsic deposits, although tyrosine nitration has been shown to occur in photoreceptor cells (Miyagi et al. 2002). However, previous studies have demonstrated that numerous structural and molecular alterations occur within human Bruch's membrane as a function of age. These changes, which disrupt the normal molecular architecture of Bruch's membrane, include: (1) structural changes in the main collagen frame work, including cross-linking and deposition of long-spaced collagen (Yamamoto and Yamashita 1989), (2) qualitative and quantitative changes in the native extracellular matrix molecules (Pauleikhoff et al. 2000), (3) deposition of abnormal extrinsic molecules including fluorescent products that accumulate in drusen (Ruberti et al. 2003), (4) macromolecular changes in the structure of Bruch's membrane, such as calcification, cracks or loss of inner layers due to inadequate basal membrane regeneration as in geographic atrophy (Feeney-Burns and Ellersieck 1985), (Grossniklaus et al. 1994), and (5) changes in the physical characteristic of Bruch's membrane, such as an age-dependent increase in trans-membrane hydraulic conductivity (Moore et al. 1995) and age-related linear decline in collagen solubility, an index of deleterious cross-linking (Karwatowski et al. 1995),
3-nitrotyrosine is known as a specific marker for inflammation-induced oxidative damage to proteins. In addition to proteins, Bruch's membrane also contains lipids, lipofuscin and carbohydrates (Hageman et al. 2001; Yasukawa et al. 2007). Lipofuscin is a mixture of autofluorescent material that accumulates in the RPE cells and is reported to photochemically generate a series of reactive oxygen species, including singlet oxygen, hydrogen peroxide, and superoxide anions (Gaillard et al. 1995; Rozanowska et al. 1998) that can enhance oxidative stress in the RPE. One of the major organic soluble chromophores in lipofuscin is A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium).
It would be desirable to be able to identify individuals with a propensity for inflammation so that an effective treatment or preventative measures can be appropriately taken for AMD.